Chimeric gaba receptor

ABSTRACT

The present invention provides an isolated GABA B  receptor protein comprising at least one GABA B R1 a  subunit and at least one GABA B R2 a  subunit, characterized in that said GABA B  receptor has one high affinity agonist binding site and one low affinity agonist binding site. In particular the isolated recombinant GABA B  receptor protein expressed by the hGABA B R1 a /GABA B R2 CHO cell line deposited at the Belgian Coordinated Collections of Microorganisms (BCCM) as CHO-K1 h-GABA-b R1 a /R2 clone on Aug. 22, 2003 with the accession number LMBP 6046CB. It is thus an object of the present invention to provide the hGABA B R1 a /GABA B R2 CHO cell line deposited at the Belgian Coordinated Collections of Microorganisms (BCCM) as CHO-K1 h-GABA-b R1 a /R2 clone on Aug. 22, 2003 with the accession number LMBP 6046CB.  
     The invention also provides the use of the aforementioned cell line in a method to identify GABA B  receptor agonists using a functional or a binding assay. In particular in a radioligand-binding assay comprising the use of radiolabeled agonists such as for example  3 H-GABA or  3 H-baclofen.  
     In a particular embodiment the present invention provides the use of the aforementioned GABA B  receptor in a method to identify a high affinity GABA B  receptor agonist using a functional or a binding assay. In particular in a radioligand-binding assay comprising the use of radiolabeled agonists such as for example  3 H-GABA or  3 H-baclofen. Alternatively, the aforementioned binding assays are performed on cellular extracts, in particular cellular membrane preparations of the aforementioned cells.

The present invention provides a novel method to identify substancesthat are agonists of GABA_(B) receptors, using a ³H-GABA binding assayin recombinant GABA_(B)R1a/R2 receptor expressing cells.

BACKGROUND OF THE INVENTION

GABA (γ-amino-butyric acid) is the most widely distributed amino acidinhibitory neurotransmitter in the central nervous system (CNS)activating two distinct families of receptors; the ionotropic GABA_(A)and GABA_(C) receptors for fast synaptic transmissions, and themetabotropic GABA_(B) receptors governing a slower synaptictransmission.

GABA_(B) receptors are members of the superfamily of seven transmembraneG-protein coupled receptors that are coupled to neuronal K⁺ or Ca²⁺channels. Presynaptic GABA_(B) receptor activation has generally beenreported to result in the inhibition of Ca²⁺ conductance, leading to adecrease in the evoked release of neurotransmitters. Post-synapticallythe major effect of GABA_(B) receptor activation is to open potassiumchannels, to generate post-synaptic inhibitory potentials.

The expression of GABA_(B) receptors is widely distributed in themammalian neuronal axis, with particularly high levels in the molecularlayer of the cerebellum, interpeduncular nucleus, frontal cortex,olfactory nuclei, thalamic nuclei, temporal cortex, raphe magnus andspinal cord. GABA_(B) receptors are also present in the peripheralnervous system, both on sensory nerves and on parasympathetic nerves.Their ability to modulate these nerves give them potential as targets indisorders of the lung, GI tract and bladder (Belley et al., 1999, Biorg.Med. Chem. 7:2697-2704).

A large number of pharmacological activities have been attributed toGABA_(B) receptor activation, such as for example, analgesia,hypothermia, catatonia, hypotension, reduction of memory consolidationand retention, and stimulation of insulin, growth hormone and glucagonrelease (see Bowery, 1989, Trends Pharmacol. Sci. 10:401-407 for areview). It is well accepted that GABA_(B) receptor agonists andantagonists are pharmacologically useful in indications such as stiffman syndrome, gastroesophogeal reflux, neuropathic pain, incontience andtreatment of cough and cocaine addiction. For example, the GABA_(B)receptor agonist baclofen has been shown to reduce transient loweresophagal sphincter relaxations (TLESR) and is accordingly useful in thetreatment of reflux as most episodes of reflux occur during TLESR.However, the current GABA_(B) receptor agonists, such as baclofen, arerelatively non-selective and show a variety of undesirable behaviouralactions such as sedation and respiratory depression. It would bedesirable to develop more GABA_(B) receptor agonists with an improvedselectivety and less of the aforementioned undesirable effects.

Current methods of drug discovery generally involve assessing thebiological activity of tens or hundreds of thousands of compounds inorder to identify a small number of those compounds having a desiredactivity against a particular target, i.e. High Throughput Screening(HTS). In a typical HTS related screen format, assays are performed inmulti-well microplates, such as 96, 384 or 1536 well plates, puttingcertain constrains to the setup of the assay to be performed includingthe availability of the source materials (i.e membrane preparations ofcells expressing the recombinant GABA_(B) receptor). HTS related screensare preferably performed at room temperature with a single measurementfor each of the compounds tested in the assay, requiring short cycletimes, with a reproducible and reliable output.

Present in vitro screens to identify compounds as agonists of theGABA_(B) receptor, either rely on natural, less abundant resources suchas binding assays in rat brain membranes or consist of functionalscreening assays, such as for example Ca²⁺ responses, c-AMP responsesand effects on Ca²⁺ and K⁺ channels performed in cells expressing arecombinant GABA_(B) receptor. In some of these functional assays theGABA_(B) receptors may be co-expressed with G-proteins, e.g. Gα16 orGqi5 or the chimeric G-protein G αq-z5, increasing G-protein coupling(Bräauner-Osborne & Krogsgaard-Larsen, 1999, Br. J. Pharmacol.128:1370-1374). However, a GABA_(B) agonist binding assay that wouldfurther reduce the HTS cycle time and the resources for biochemicalssuch as recombinant proteins, is currently unavailable.

The present invention describes the development of a Chinese HamsterOvary (CHO) cell line co-expressing the human GABA_(B) receptor subunitsGABA_(B)R1a and GABA_(B)R2, which were surprisingly found to demonstrateagonist binding in radioligand binding experiments. In addition, thepresent inventors demonstrated that the hGABA_(B)R1a/GABA_(B)R2 CHO cellline has one high affinity and one low affinity agonist binding site inthe recombinant expressed GABA_(B) receptor. Hence thehGABA_(B)R1a/GABA_(B)R2 CHO cell line provided by the present inventionnot only allows compound screening, but also provides a useful tool tocharacterize the nature of the compound-receptor interaction.

SUMMARY OF THE INVENTION

The present invention provides an isolated GABA_(B) receptor proteincomprising at least one GABA_(B)R1a subunit and at least one GABA_(B)R2subunit, characterized in that said GABA_(B) receptor has one highaffinity agonist binding site and one low affinity agonist binding site.In particular the isolated recombinant GABA_(B) receptor proteinexpressed by the hGABA_(B)R1a/GABA_(B)R2 CHO cell line deposited at theBelgian Coordinated Collections of Microorganisms (BCCM) as CHO-K1h-GABA-b R1a/R2 clone on Aug. 22, 2003 with the accession number LMBP6046CB. It is thus an object of the present invention to provide thehGABA_(B)R1a/GABA_(B)R2 CHO cell line deposited at the BelgianCoordinated Collections of Microorganisms (BCCM) as CHO-K1 h-GABA-bR1a/R2 clone on Aug. 22, 2003 with the accession number LMBP 6046CB.

The invention also provides the use of the aforementioned cell line in amethod to identify GABA_(B) receptor agonists using a functional or abinding assay. In particular in a radioligand-binding assay comprisingthe use of radiolabeled agonists such as for example ³H-GABA or³H-baclofen.

The invention further provides a method to identify GABA_(B) receptoragonists, comprising contacting the aforementioned cell line with a testcompound and measuring the binding of said test compound to the GABA_(B)receptor. In particular the method consists of a radioligand bindingassay, comprising exposing the aforementioned cells to a labelledagonist of GABA_(B) in the presence and absence of the test compound andmeasure the binding of the labelled ligand to the cells according to theinvention, where if the amount of binding of the labelled ligand is lessin the presence of the test compound, then the compound is a potentialagonist of the GABA_(B) receptor.

It is also an object of the present invention to provide a method toidentify a high affinity GABA_(B) receptor agonist, said methodcomprising contacting the aforementioned cells with the radiolabeledagonist selected from the group consisting of GABA, baclofen and3-aminopropylphosphinic acid (3-APPA a.k.a APMPA), in the presence andabsence of the test compound and measure the binding of the labelledligand to the cells according to the invention, where if the amount ofbinding of the labelled ligand to the high affinity binding site is lessin the presence of the test compound, then the compound is a potentialhigh affinity agonist of the GABA_(B) receptor.

Alternatively, the aforementioned binding assays are performed oncellular extracts, in particular cellular membrane preparations of thehGABA_(B)R1a/GABA_(B)R2 CHO cell line deposited at the BelgianCoordinated Collections of Microorganisms (BCCM) as CHO-K1 h-GABA-bR1a/R2 clone on Aug. 22, 2003 with the accession number LMBP 6046CB.

In another embodiment the present invention provides a method toidentify a GABA_(B) receptor agonist, said method comprising contactingthe aforementioned cell line with a compound to be tested and determinewhether the compound activates a GABA_(B) receptor functional responsein said cells. In particular the functional response consists ofmodulation of the activity of ion channels or of intracellularmessengers as explained hereinafter.

This and further aspects of the present invention will be discussed inmore detail hereinafter.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 GTPγ35S-binding upon stimulation of membranes by GABA expressedas the percentage of maximal GABA stimulation, in the presence andabsence of the positive allosteric modulator CGP7930>

FIG. 2 Displacement of ³H-GABA by agonists (baclofen, GABA & APMPA) andantagonists (SCH50911 & CGP54626)

FIG. 3 Reproducible agonist IC₅₀ values (n=5) independent of membranepreparations.

FIG. 4 Two sided ³H-GABA agonist binding curve in the presence orabsence of 10 μM CGP54626 (a) or JNJ 4309747-AAD (b).

DETAILED DESCRIPTION

For the purposes of describing the present invention: GABA_(B)R1a or hGABA_(B)R1a as used herein refers to the human GABA_(B) receptor subunitknown as GABA_(B)R1a in Kaupmann et al, 1998, Proc. Natl. Acad. Sci. USA95:14991-14996, the amino acid sequence (SEQ ID No.:2) of which can befound at GenBank Accession no. AJ225028, as well as to its mammalianorthologs. GABA_(B)R1a also refers to other GABA_(B) receptor subunitsthat have minor changes in amino acid sequence from those describedhereinbefore, provided those other GABA_(B) receptor subunits havesubstantially the same biological activity as the subunits describedhereinbefore. A GABA_(B)R1a subunit has substantially the samebiological activity if it has an amino acid sequence that is at least80% identical to, preferably at least 95% identical to, more preferablyat least 97% identical to, and most preferably at least 99% identical toSEQ ID No.: 2 and has a Kd or EC50 for GABA, GABA_(B) receptor agonistssuch as for example baclofen and gabapentin or GABA_(B) receptorantagonists such as for example CGP54626A, SCH 50911, saclofen andphaclofen, that is no more than 5-fold greater than the Kd or EC50 of anative GABA_(B) receptor for GABA or the same GABA_(B) receptor agonistor GABA_(B) receptor antagonist.

GABA_(B)R2 as used herein refers to the human GABA_(B) receptor subunitknown as GABA_(B)R2 in White et al., 1998, Nature 396:679-682, the aminoacid sequence (Seq Id NO.: 4) of which can be found at GenBank accessionno. AF058795 as well as to its mammalian orthologs. GABA_(B)R2 alsorefers to other GABA_(B) receptor subunits that have minor changes inamino acid sequence from those described hereinbefore, provided thoseother GABA_(B) receptor subunits have substantially the same biologicalactivity as the subunits described hereinbefore. A GABA_(B)R2 subunithas substantially the same biological activity if it has an amino acidsequence that is at least 80% identical to, preferably at least 95%identical to, more preferably at least 97% identical to, and mostpreferably at least 99% identical to SEQ BD No.: 4 and has incombination with a GABA_(B)R1 subunit a Kd or EC50 for GABA, GABA_(B)receptor agonists such as for example baclofen and gabapentin orGABA_(B) receptor antagonists such as for example CGP54626A, SCH 50911,saclofen and phaclofen, that is no more than 5-fold greater than the Kdor EC50 of a native GABA_(B) receptor for GABA or the same GABA_(B)receptor agonist or GABA_(B) receptor antagonist.

The Kd and EC50 values of the native GABA_(B) receptor is determinedusing the methods known to a person skilled in the art, in particularusing competition binding studies on tissue preparations such as forexample described in Cross & Horton, 1987 Eur.J.Pharmacol. 141(1):159-162. Briefly, crude synaptic membranes are prepared byhomogenisation of whole brain, centrifugation (30 000×g, 20 min.) andextensive washing. Total binding is measured by incubation of themembranes with ³H-GABA or ³H-baclofen, while non-specific binding ismeasured in the presence of 100 μM baclofen. Upon removal of unboundligand by filtration, filters are counted in a β-counter or a TopcountHarvester (Packhard). For competition experiments the binding occurs inthe presence of increasing concentration of unlabeled compound.

It is thus an object of the present invention to provide an isolatedGABA_(B) receptor protein formed by at least one GABA_(B)R1a and atleast one GABA_(B)R2 subunit further characterized in that said isolatedGABA_(B) has both a high and a low affinity agonist binding site. In afurther embodiment this isolated GABA_(B) receptor is a functionalGABA_(B) receptor expressed by a cell, wherein said cell does notnormally express the GABA_(B) receptor. Suitable cells which arecommercially available, include but are not limited to L-cells, HEK-293cells, COS cells, CHO cells, HeLa cells and MRC cells, in particular CHOcells wherein the GABA_(B) receptor protein comprises at least oneGABA_(B)R1a subunit encoded by the oligonucleotide sequence consistingof SEQ ID No.1 and at least one GABA_(B)R2 subunit encoded by theoligonucleotide sequence consisting of SEQ ID No.3. In a more particularembodiment the isolated GABA_(B) receptor according to the invention,consists of the receptor protein expressed by thehGABA_(B)R1a/GABA_(B)R2 CHO cell line deposited at the BelgianCoordinated Collections of Microorganisms (BCCM) as CHO-K1 h-GABA-bR1a/R2 clone 20 on Aug. 22, 2003 with accession number LMBP 6046CB.

“Functional GABA_(B) receptor”refers to a GABA_(B) receptor formed byco-expression of GABA_(B)R2 and GABA_(B)R1a in a cell, wherein said celldoes not normally express the GABA_(B) receptor, most preferablyresulting in a heterodimer of GABA_(B)R2 and GABA_(B)R1a, where thefunctional GABA_(B) receptor mediates at least one functional responsewhen exposed to the GABA_(B) receptor agonist GABA. Examples offunctional responses are: pigment aggregation in Xenopus melanophores,negative modulation of cAMP levels, coupling to inwardly rectifyingpotassium channels, mediation of late inhibitory postsynaptic potentialsin neurons, increases in potassium conductance, decreases in calciumconductance, MAPKinase activation, extracellular pH acidification, andother functional responses typical of G-protein coupled receptors. Oneskilled in the art would be familiar with a variety of methods ofmeasuring the functional responses of G-protein coupled receptors suchas the GABA_(B) receptor (see, e. g., Lerner, 1994, Trends Neurosci.17:142-146 [changes in pigment distribution in melanophore cells]; Yokomizoet al., 1997, Nature 387: 620-624 [changes in cAMP or calciumconcentration; chemotaxis]; Howard et al., 1996, Science 273: 974-977[changes in membrane currents in Xenopus oocytes]; McKee et al., 1997,Mol. Endocrinol. 11: 415-423 [changes in calcium concentration measuredusing the aequorin assay]; Offermanns & Simon, 1995, J. Biol. Chem. 270:15175-15180 [changes in inositol phosphate levels]). Depending upon thecells in which heteromers of GABA_(B)R1a and GABA_(B)R2 are expressed,and thus the G-proteins with which the functional GABA_(B) receptor thusformed is coupled, certain of such methods may be appropriate formeasuring the functional responses of such functional GABA_(B)receptors. It is well within the competence of one skilled in the art toselect the appropriate method of measuring functional responses for agiven experimental system.

The term “compound”, “test compound”, “agent” or “candidate agent” asused herein can be any type of molecule, including for example, apeptide, a polynucleotide, or a small molecule that one whishes toexamine for their activity as GABA_(B) receptor agonist, and whereinsaid agent may provide a therapeutic advantage to the subject receivingit. The candidate agents can be administered to an individual by variousroutes, including, for example, orally or parenterally, such asintravenously, intramuscularly, subcutaneously, intraorbitally,intracapsularly, intraperitoneally, intrarectally, intracisternally orby passive or facilitated absorption through the skin, using for examplea skin patch or transdermal iontophoresis, respectively. Furthermore thecompound can be administered by injection, intubation or topically, thelatter of which can be passive, for example, by direct application of anointment, or active, for example, using a nasal spray or inhalant, inwhich case one component of the composition is an appropriatepropellant. The route of administration of the compound will depend, inpart, on the chemical structure of the compound. Peptides andpolynucleotides, for example, are not particular useful whenadministered orally because they can be degraded in the digective tract.However, methods for chemically modifying peptides, for examplerendering them less susceptible to degradation are well know and includefor example, the use of D-amino acids, the use of domains based onpeptidomimetics, or the use of a peptoid such as a vinylogous peptoid.

The agent used in the screening method may be used in a pharmaceuticallyacceptable carrier. See, e.g., Remington's Pharmaceutical Sciences,latest edition, by E.W. Martin Mack Pub. Co., Easton, Pa., whichdiscloses typical carriers and conventional methods of preparingpharmaceutical compositions that may be used in conjunction with thepreparation of formulations of the agents and which is incorporated byreference herein.

Cells

As already outlined above, the present invention provides a cell linestably transfected with expression vectors that direct the expression ofthe GABA_(B) receptor subunits GABA_(B)R1a and GABA_(B)R2 as definedhereinbefore. In particular CHO cells transfected with said expressionvectors. Such expression vectors are routinely constructed in the art ofmolecular biology and may involve the use of plasmid DNA and appropriateinitiators, promoters, enhancers and other elements, which may benecessary, and which are positioned in the correct orientation, in orderto allow for protein expression. Generally, any system or vectorsuitable to maintain, propagate or express polynucleotides to produce apolypeptide in a host may be used. The appropriate nucleotide sequence,i.e. the polynucleotide sequences encoding either the human GABA_(B)R1aor GABA_(B)R2 subunit as defined hereinbefore, may be inserted into anexpression system by any of a variety of well-known and routinetechniques such as for example those set forth in Current Protocols inMolecular Biology, Ausbel et al. eds., John Wiley & Sons, 1997.

In a particular embodiment the CHO cells according to the invention arecotransfected with the commercially available expression vectorspcDNA3.1 comprising the polynucleotide sequences encoding for humanGABA_(B)R1a (SEQ ID No.: 1) and human GABA_(B)R2 (SEQ ID No.: 3)respectively. More preferably the present invention provides ahGABA_(B)R1a/GABA_(B)R2 CHO cell line deposited at the BelgianCoordinated Collections of Microorganisms (BCCM) as CHO-K1 h-GABA-bR1a/R2 clone 20 on Aug. 22, 2003 with the accession number LMBP 6046CB.This cell line is characterized in that the functional GABA_(B) receptorin this CHO cell line has both a low and a high affinity binding sitefor GABA_(B) receptor agonist. Using the cell line according to theinvention, will not only allow compound screening, but also provides auseful tool for the characterization of the nature of thecompound-receptor interaction, i.e. does it interact with the low orhigh affinity agonist binding site of the GABA_(B) receptor.

For further details in relation to the preparation of nucleic acidconstructs, mutagenesis, sequencing, introduction of DNA into cells andgene expression, and analysis of proteins, see for example, MolecularCloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, ColdSpring Harbor Laboratory Press.

Assays

The present invention also provides an assay for a compound capable ofinteracting with the functional GABA_(B) receptor of the presentinvention, which assay comprises: providing the GABA_(B) receptorexpressed by the hGABA_(B)R1a/GABA_(B)R2 CHO cell line of the presentinvention, contacting said receptor with a putative binding compound;and determining whether said compound is able to interact with saidreceptor.

In one embodiment of the assay, the receptor or subunits of the receptormay be employed in a binding assay. Binding assays may be competitive ornon-competitive. Such an assay can accommodate the rapid screening of alarge number of compounds to determine which compounds, if any, arecapable of binding to the polypeptides.

Within this context, the present invention provides a method to identifywhether a test compound binds to an isolated GABA_(B) receptor proteinof the present invention, and is thus a potential agonist or antagonistof the GABA_(B) receptor, said method comprising;

a) contacting cells expressing a functional GABA_(B) receptor, whereinsuch cells do not normally express the GABA_(B) receptor, with the testcompound in the presence and absence of a compound known to bind theGABA_(B) receptor, and

b) determine the binding of the test compound to the GABA_(B) receptorusing the compound known to bind to the GABA_(B) receptor as areference.

Binding of the test compound or of the compound known to bind to theGABA_(B) receptor, hereinafter also referred to as reference compound,is assessed using art-known methods for the study of protein-ligandinteractions. For example, such binding can be measured by employing alabeled substance or reference compound. The test compound or referencecompound can be labeled in any convenient manner known in the art, e.g.radioactively, fluorescently or enzymatically. In a particularembodiment of the aforementioned method, the compound known to bind tothe GABA_(B) receptor, a.k.a. the reference compound is detectablylabeled, and said label is used to determine the binding of the testcompound to the GABA_(B) receptor. Said reference compound being labeledusing a radiolabel, a fluorescent label or an enzymatic label, morepreferably a radiolabel. In a more particular embodiment, the presentinvention provides a method to identify whether a test compound binds toan isolated GABA_(B) receptor protein, said method comprising the use ofa compound known to bind to the GABA_(B) receptor, wherein saidreference compound is selected from the group consisting of ³H-GABA,³H-baclofen, ³H-3-APPA, ³H-CGP542626 and ³H-SCH50911.

Subsequently, more detailed assays can be carried out with thosecompounds found to bind, to further determine whether such compounds actas agonists or antagonists of the polypeptides of the invention.

Thus, in a further embodiment the present invention provides a method toidentify GABA_(B) receptor agonists said method comprising,

-   a) exposing cells expressing a functional GABA_(B) receptor, wherein    such cells do not normally express the GABA_(B) receptor, to a    labeled agonists of GABA_(B) in the presence and absence of the test    compound, and-   b) determine the binding of the labeled agonist to said cells,    where if the amount of binding of the labeled agonist is less in the    presence of the test compound, then the compound is a potential    agonist of the GABA_(B) receptor. As already specified for the    general binding assay above, the binding of the GABA_(B) receptor    agonists is assessed using art-known methods for the study of    protein-ligand interactions. The label is generally selected from a    radioactive label, a fluorescent label or an enzymatic label, in    particular a radiolabel wherein the agonist is selected from the    group consisting of ³H-GABA, ³H-baclofen and ³H-3-APPA.

Similarly, the present invention provides a method to identify GABA_(B)receptor antagonists said method comprising,

-   a) exposing cells expressing a functional GABA_(B) receptor, wherein    said cells do not normally express the GABA_(B) receptor, to a    labeled antagonist of GABA_(B) in the presence and absence of the    test compound, and-   b) determine the binding of the labeled antagonist to said cells,

where if the amount of binding of the labeled antagonist is less in thepresence of the test compound, then the compound is a potentialantagonist of the GABA_(B) receptor. As already specified for thegeneral binding assay above, the binding of the GABA_(B) receptorantagonists is assessed using art-known methods for the study ofprotein-ligand interactions. The label is generally selected from aradioactive label, a fluorescent label or an enzymatic label, inparticular a radiolabel wherein the antagonist is selected from thegroup consisting of ³H-CGP542626 and ³H-SCH50911.

In an alternative embodiment of the present invention, theaforementioned binding assays are performed on a cellular composition,i.e a cellular extract, a cell fraction or cell organels comprising aGABA_(B) receptor as defined hereinbefore. More in particular, theaforementioned binding assays are performed on a cellular composition,i.e. a cellular extract, a cell fraction or cell organels comprising aGABA_(B) receptor as defined hereinbefore, wherein said cellularcomposition, i.e. cellular extract, cell fraction or cell organels, isobtained from cells expressing a functional GABA_(B) receptor, whereinsaid cells do not normally express the GABA_(B) receptor. Morepreferably, the cellular composition, i.e. cellular extract, cellfraction or cell organels, is obtained from the hGABA_(B)R1a/GABA_(B)R2CHO cell line deposited at the Belgian Coordinated Collections ofMicroorganisms (BCCM) as CHO-K1 h-GABA-b R1a/R2 clone 20 on Aug. 22,2003 with the accession number LMBP 6046CB.

It is accordingly, an object of the present invention to provide amethod for identifying a compound as a GABA_(B) receptor agonist orantagonist, said method comprising;

-   a) administering the compound to a cellular composition of cells    expressing a functional GABA_(B) receptor, wherein said cells do not    normally express the GABA_(B) receptor, in the presence of a    detectably labeled agonist or antagonist of the GABA_(B) receptor;    and-   b) determine the binding of the labeled agonist or antagonist to    said cellular composition,

where if the amount of binding of the labeled agonist or antagonist isless in the presence of the test compound, then the compound is apotential agonist respectively antagonist of the GABA_(B) receptor.

As already specified for the general binding assay above, the binding ofthe GABA_(B) receptor agonist or antagonist is assessed using art-knownmethods for the study of protein-ligand interactions. The label isgenerally selected from a radioactive label, a fluorescent label or anenzymatic label, in particular a radiolabel wherein the agonist isselected from the group consisting of ³H-GABA, ³H-baclofen and 3H-3-APPAand the antagonist is selected from the group consisting of ³H-CGP542626and ³H-SCH50911. In a more specific embodiment the aforementionedbinding assays are performed on a cellular composition consisting of themembrane fraction of cells according to the invention, in particular onmembrane fractions of the hGABA_(B)R1a/GABA_(B)R2 CHO cell linedeposited at the Belgian Coordinated Collections of Microorganisms(BCCM) as CHO-K1 h-GABA-b R1a/R2 clone 20 on Aug. 22, 2003 with theaccession number LMBP 6046CB, using one or more of the aforementionedradiolabeled agonsist and/or antagonists.

In a further embodiment the present invention provides a functionalassay for identifying compounds that modulate the GABA_(B)-receporactivity in the cells according to the invention. Such an assay isconducted using the cells of the present invention, i.e. cotranfectedwith the human GABA_(B)R1a and human GABA_(B)R2 subunits. The cells arecontacted with at least one reference compound wherein the ability ofsaid compound to modulate the GABA_(B)-receptor activity is known.Thereafter, the cells are contacted with a test compound and determinedwhether said test compound modulates the activity of the GABA_(B)receptor compared to the reference compound. A “reference compound” asused herein refers to a compound that is known to bind and/or tomodulate the GABA_(B) receptor activity.

A compound or a signal that “modulates the activity” of a polypeptide ofthe invention refers to a compound or a signal that alters the activityof the polypeptide so that it behaves differently in the presence of thecompound or signal than in the absence of the compound or signal.Compounds affecting modulation include agonists and antagonists. Anagonist of the GABA_(B) receptor encompasses a compound such as GABA,baclofen and 3-APPA which activates GABA_(B) receptor function.Alternatively, an antagonist includes a compound that interferes withGABA_(B) receptor function. Typically, the effect of an antagonist isobserved as a blocking of agonist-induced receptor activation.Antagonists include competitive as well as non-competitive antagonists.A competitive antagonist (or competitive blocker) interacts with or nearthe site specific for agonist binding. A noncompetitive antagonist orblocker inactivates the function of the receptor by interacting with asite other than the agonist interaction site.

In one embodiment the present invention provides a method foridentifying compounds that have the capability to modulate GABA_(B)receptor activity, said method comprising;

a) contacting cells expressing a functional GABA_(B) receptor, whereinsaid cells do not normally express a functional GABA_(B) receptor, withat least one reference compound, under conditions permitting theactivation of the GABA_(B) receptor;

-   b) contacting the cells of step a) with a test compound, under    conditions permitting the activation of the GABA_(B) receptor, and-   c) determine whether said test compound modulates the GABA_(B)    receptor activity compared to the reference compound.

Methods to determine the capability of a compound to modulate theGABA_(B) receptor activity are based on the variety of assays availableto determine the functional response of G-protein coupled receptors (seeabove) and in particular on assays to determine the changes in potassiumcurrents, changes in calcium concentration, changes in cAMP and changesin GTPγS binding. Conditions permitting the activation of the GABA_(B)receptor generally known in the art, for example in case of antagonistscreening these conditions comprise the presence of a GABA_(B) receptoragonist in the assay system. Typical GABA_(B) receptor agonists used inthese activity assays are GABA, baclofen or 3-APPA. More particular inthe GTPγS assay as outlined herein below, GABA is used to activate theGABA_(B) receptor in order to assess the capability of a test compoundto inactivate the GABA_(B) receptor protein.

In the aforementioned assay an increase of GTPγS binding in the presenceof the test compound is an indication that the compound activates theGABA_(B) receptor activity, and accordingly that said test compound is apotential agonist of the GABA_(B) receptor protein. A decrease of GTPγSbinding in the presence of the test compound is an indication that thecompound inactivates the GABA_(B) receptor protein and accordingly thatsaid test compound is a potential antagonist of the GABA_(B) receptorprotein.

Particularly preferred types of assays include binding assays andfunctional assays which may be performed as follows:

Binding Assays

Over-expression of the GABA_(B) receptor expressed by thehGABA_(B)R1a/GABA_(B)R2 CHO cell line of the present invention may beused to produce membrane preparations bearing said receptor (referred toin this section as GABA_(B) binding receptor for convenience) for ligandbinding studies. These membrane preparations can be used in conventionalfilter-binding assays (eg. Using Brandel filter assay equipment) or inhigh throughput Scintillation Proximity type binding assays (SPA andCytostar-T flashplate technology; Amersham Pharmacia Biotech) to detectbinding of radio-labelled GABA_(B) ligands (including ³H-GABA,³H-baclofen, ³H-3-APPA, ³H-CGP542626, ³H-SCH50911) and displacement ofsuch radio-ligands by competitors for the binding site. Radioactivitycan be measured with Packard Topcount, or similar instrumentation,capable of making rapid measurements from 96-, 384-, 1536-microtitrewell formats. SPA/Cytostar-T technology is particularly amenable to highthroughput screening and therefore this technology is suitable to use asa screen for compounds able to displace standard ligands.

Another approach to study binding of ligands to GABA_(B) bindingreceptor protein in an environment approximating the native situationmakes use of a surface plasmon resonance effect exploited by the Biacoreinstrument (Biacore). GABA_(B) binding receptor in membrane preparationsor whole cells could be attached to the biosensor chip of a Biacore andbinding of ligands examined in the presence and absence of compounds toidentify competitors of the binding site.

Functional Assays

Since GABA_(B) receptors belong to the family G-protein coupledreceptors that are coupled to GIRK (inward rectifying potassiumchannels), potassium ion flux should result on activation of thesereceptors. This flux of ions may be measured in real time using avariety of techniques to determine the agonistic or antagonistic effectsof particular compounds. Therefore, recombinant GABA_(B) bindingreceptor proteins expressed in the cell lines of the present inventioncan be characterised using whole cell and single channelelectrophysiology to determine the mechanism of action of compounds ofinterest. Electrophysiological screening, for compounds active atGABA_(B) binding receptor proteins, may be performed using conventionalelectrophysiological techniques and when they become available, novelhigh throughput methods currently under development.

Given the presynaptic effect of GABA_(B) receptor activation on Ca²⁺channels, in an alternative functional screen the modulatory effect of acompound is assessed through the changes in intracellular calcium.Calcium fluxes are measurable using several ion-sensitive fluorescentdyes, including fluo-3, fluo4, fluo-5N, fura red and other similarprobes from suppliers including Molecular Probes. The inhibition ofcalcium influx as a result of GABA_(B) receptor activation can thus becharacterised in real time, using fluorometric and fluorescence imagingtechniques, including fluorescence microscopy with or without laserconfocal methods combined with image analysis algorithms.

Another approach is a high throughput screening assay for compoundsactive as either agonists or modulators which affect calcium transients.This assay is based around an instrument called a Fluorescence ImagingPlate Reader ((FLIPR®), Molecular Devices Corporation). In its mostcommon configuration, it excites and measures fluorescence emitted byfluorescein-based dyes. It uses an argon-ion laser to produce high powerexcitation at 488 nm of a fluorophore, a system of optics to rapidlyscan the over the bottom of a 96-/384-well plate and a sensitive, cooledCCD camera to capture the emitted fluorescence. It also contains a96-/384-well pipetting head allowing the instrument to deliver solutionsof test agents into the wells of a 96-/384-well plate. The FLIPR assayis designed to measure fluorescence signals from populations of cellsbefore, during and after addition of compounds, in real time, from all96-/384-wells simultaneously. The FLIPR assay may be used to screen forand characterise compounds functionally active at thehGABA_(B)R1a/GABA_(B)R2 CHO cell line.

A high throughput screening assay, specifically usefull to identifyGABA_(B) agonists could consist of an arrangement whereinhGABA_(B)R1a/GABA_(B)R2 CHO cells, are loaded with an appropriatefluorescent dye, incubated with a test compound and after sufficienttime to allow interaction (8-24 hours, typically 12-24 hours, inparticular 24 hours.) the change in relative fluorescence units measuredusing an automated fluorescence plate reader such as FLIPR or AscentFluoroskan (commercially available from Thermo Labsystems, Brussel,Belgium).

In a further embodiment the functional assay is based on the change inGTPγS binding to the GABA_(B) binding receptor. In particular using acompetion bindig assay to determine the displacement of radiolabelledGTPγS. In general, this method to identify GABA_(B)-receptor agonistscomprises preparing a membrane fraction from cells expressing thehGABA_(B)R1a/GABA_(B)R2 heterodimer af the present invention, contactingsaid membrane preparations with the compound to be tested in thepresence of radiolabelled GTPγS, under conditions permitting theactivation of the GABA_(B) receptor, and detecting GTPγS binding to themembrane fraction. An increase in GTPγS binding in the presence of thecompound is an indication that the compound activates thehGABA_(B)R1a/GABA_(B)R2 receptor. A decrease in GTPγS binding in thepresence of the compound is an indication that the compound inactivatesthe hGABA_(B)R1a/GABA_(B)R2 receptor. Preferably this GTPγS bindingassay is performed on membrane fractions obtained from thehGABA_(B)R1a/GABA_(B)R2 CHO cell line deposited at the BelgianCoordinated Collections of Microorganisms (3CCM) as CHO-K1 h-GABA-bR1a/R2 clone 20 on Aug. 22, 2003 with the accession number LMBP 6046CB.Further, the conditions permitting the activation of the GABA_(B)receptor comprise the presence of a GABA_(B) receptor agonist, such asfor example GABA, baclofen and 3-APPA in the assay system. In particularGABA.

This and other functional screening assays will be provided in theexamples hereinafter.

GABA_(B) receptor agonists

In a further aspect the present invention provides GABA_(B) receptoragonists identified using one of the aforementioned screening assayswherein said GABA_(B) receptor agonists are represented by the compoundsof formula (I)

the N-oxide forms, the pharmaceutically acceptable addition salts andthe stereochemically isomeric forms thereof, wherein

-   -   =Z¹-Z²=Z³-Z⁴=represents a divalent radical selected from the        group consisting of        -   ═N—CH═CH—N═ (a), ═N—CH═N—CH═ (b), ═CH—N═CH—N═ (c)            ═CH—CH═CH—CH═ (d), ═N—CH═CH—CH═ (e), ═CH—N═CH—CH═ (f),            ═CH—CH═N—CH═ (g) and ═CH—CH═CH—N═ (h);    -   R¹ represents hydrogen, halo, hydroxyl, cyano, C₁₋₆alkyl, CF₃,        amino or mono- or di(C₁₋₄alkyl)amino;    -   R² represents hydrogen, C₁₋₆alkyl or hydroxycarbonyl-C₁₋₆alkyl-.

In particular those compounds of formula (I) wherein one or more of thefollowing restrictions apply;

-   -   (i) =Z¹-Z²=z³-Z⁴=represents a divalent radical selected from the        group consisting of        -   ═N—CH═CH—N═ (a), ═N—CH═N-CH═ (b), ═CH—N═CH—N═ (c) and            ═CH—CH═CH—CH═ (d);    -   (ii) R¹ represents halo, amino or mono- or di(C₁₋₄alkyl)amino;    -   (iii) R² represents butyric acid

Also of interest are those compounds of formula (I) wherein;

-   -   (i) R¹ is attached at position Z¹; and/or    -   (ii) =Z¹-Z²=Z³ -Z⁴=represents (a), (b) or (d), more preferably        =Z¹-Z²=Z³-Z⁴=represents (d).

As used in the foregoing definitions and hereinafter, halo is generic tofluoro, chloro, bromo and iodo; C₁₋₄alkyl defines straight and branchedchain saturated hydrocarbon radicals having from 1 to 4 carbon atomssuch as, for example, methyl, ethyl, propyl, butyl, 1-methylethyl,2-methylpropyl, 2,2-dimethylethyl and the like; C₁₋₆alkyl definesstraight and branched chain saturated hydrocarbon radicals having from 1to 6 carbon atoms such as, for example, pentyl, hexyl, 3-methylnutyl,2-methylpentyl and the like.

The pharmaceutically acceptable addition salts as mentioned hereinaboveare meant to comprise the therapeutically active non-toxic acid additionsalt forms, which the compounds of formula (I), are able to form. Thelatter can conveniently be obtained by treating the base form with suchappropriate acid. Appropriate acids comprise, for example, inorganicacids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid;sulfuric; nitric; phosphoric and the like acids; or organic acids suchas, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic,oxalic, malonic, succinic (i.e. butanedioic acid), maleic, fumaric,malic, tartaric, citric, methanesulfonic, ethanesulfonic,benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic,p-aminosalicylic, pamoic and the like acids.

The pharmaceutically acceptable addition salts as mentioned hereinaboveare meant to comprise the therapeutically active non-toxic base additionsalt forms which the compounds of formula (I), are able to form.Examples of such base addition salt forms are, for example, the sodium,potassium, calcium salts, and also the salts with pharmaceuticallyacceptable amines such as, for example, ammonia, alkylamines,benzathine, N-methyl-D-glucamine, hydrabamine, amino acids, e.g.arginine, lysine.

Conversely said salt forms can be converted by treatment with anappropriate base or acid into the free acid or base form.

The term addition salt as used hereinabove also comprises the solvateswhich the compounds of formula (I), as well as the salts thereof, areable to form. Such solvates are for example hydrates, alcoholates andthe like.

The term stereochemically isomeric forms as used hereinbefore definesthe possible different isomeric as well as conformational forms whichthe compounds of formula (I), may possess. Unless otherwise mentioned orindicated, the chemical designation of compounds denotes the mixture ofall possible stereochemically and conformationally isomeric forms, saidmixtures containing all diastereomers, enantiomers and/or conformers ofthe basic molecular structure. All stereochemically isomeric forms ofthe compounds of formula (I), both in pure form or in admixture witheach other are intended to be embraced within the scope of the presentinvention.

The N-oxide forms of the compounds of formula (I), are meant to comprisethose compounds of formula (I) wherein one or several nitrogen atoms areoxidized to the so-called N-oxide.

The 7,8-dihydro-phenothiazine derivatives of the present invention aregenerally prepared as described by Nemeryuk M. P. et al.,Khimiko-Farmatsevticheskii Zhumal (1985), 19(8), 964-968. In brief, theknown ortho-amino substituted (hetero)arene-thiols (II), are condensedwith an appropriate 2-bromo-5,5-dimethyl-3-oxo-cyclohex-1-enylaminoderivative (III), by heating the two reactants in a suitable solvent,such as ethanol or N-methylpyrrolidone. Standard work-up andpurification gives the desired products of formula I (Scheme 1).

Wherein =Z¹-Z²=Z³-Z⁴=, R¹ and R² are defined as for the compounds offormula (I) hereinbefore.

The appropriate 2-bromo-5,5-dimethyl-3-oxo-cyclohex-1-enylaminoderivatives (III) can generally be obtained by amination of5,5-dimethyl-1,3-cyclohexanedione with the appropriate amine of generalformula (IV) under art known amination conditions, followed bybromination with N-bromosuccinimlide (Scheme 2).

Wherein R² is defined as for the compounds of formula (I) hereinbefore.

For those compounds of formula (I) where R² represents butyric acid,hereinafter referred to as the compounds of formula (I′), the compoundsare obtained by condensing the ortho-amino substituted(hetero)arene-thiol (II) with4-(2-bromo-5,5-dimethyl-3-oxo-cyclohex-1-enylamino)-butyric acid or anester derivative such as a t-butylester (V) using art known conditions,such as for example by heating the two reactants in a suitable solvent,such as ethanol or N-methylpyrrolidone. Standard work-up andpurification gives the desired products, or the ester derivative, whichcan be hydrolyzed under acidic or basic conditions to give the requiredbutyric acids (I′) (Scheme 3).

Further examples for the synthesis of compounds of formula (I) using theabove mentioned synthesis method is provided in the experimental parthereinafter.

Where necessary or desired, any one or more of the following furthersteps in any order may be performed:

-   -   (i) removing any remaining protecting group(s);    -   (ii) converting a compound of formula (I) or a protected form        thereof into a further compound of formula (I) or a protected        form thereof;    -   (iii) converting a compound of formula (I) or a protected form        thereof into a N-oxide, a salt, a quaternary amine or a solvate        of a compound of formula (I) or a protected form thereof;    -   (iv) converting a N-oxide, a salt, a quaternary amine or a        solvate of a compound of formula (I) or a protected form thereof        into a compound of formula (I) or a protected form thereof;    -   (v) converting a N-oxide, a salt, a quaternary amine or a        solvate of a compound of formula (I) or a protected form thereof        into another N-oxide, a pharmaceutically acceptable addition        salt a quaternary amine or a solvate of a compound of        formula (I) or a protected form thereof.

It will be appreciated by those skilled in the art that in the processesdescribed above the functional groups of intermediate compounds may needto be blocked by protecting groups.

Functional groups which it is desirable to protect include hydroxy,amino and carboxylic acid. Suitable protecting groups for hydroxyinclude trialkylsilyl groups (e.g. tert-butyldimethylsilyl,tert-butyldiphenylsilyl or trimethylsilyl), benzyl andtetrahydro-pyranyl. Suitable protecting groups for amino includetert-butyloxycarbonyl or benzyloxycarbonyl. Suitable protecting groupsfor carboxylic acid include C₁₋₆)alkyl or benzyl esters.

The protection and deprotection of functional groups may take placebefore or after a reaction step.

The use of protecting groups is fully described in ‘Protective Groups inOrganic Synthesis’ 3^(rd) edition, T W Greene & P G M Wutz, John Wiley &Sons Inc. (June 1999).

Additionally, the N-atoms in compounds of formula (I) can be methylatedby art-known methods using CH₃—I in a suitable solvent such as, forexample 2-propanone, tetrahydrofuran or dimethylformamide.

Some of the intermediates and starting materials as used in the reactionprocedures mentioned hereinabove are known compounds and may becommercially available or may be prepared according to art-knownprocedures.

Method of Treatment

The present invention also provides the use of a compound identified asa GABA_(B) receptor activity modulator, using one of the aforementionedassays, in particular the compounds of formula (I) as describedhereinbefore, in the manufacture of a medicament for the treatment anindication such as stiff man syndrome, gastroesophogeal reflux,neuropathic pain, incontinence and treatment of cough and cocaineaddiction. In particular for use in the manufacture of a medicament toreduce transient lower esophagal sphincter relaxations (TLESR). It isthus an object of the present invention to provide a method for thetreatment of a warm-blooded animal, for example, a mammal includinghumans, suffering from an indication such as stiff man syndrome,gastroesophogeal reflux, neuropathic pain, incontinence and treatment ofcough and cocaine addiction, in particular TLESR.

Said method comprising administering to a warm-blooded animal in needthereof an effective amount of a compound identified as a GABA_(B)receptor modulator using a method according to the invention. Inparticular the systemic or topical administration of an effective amountof a compound according to the invention, to warm-blooded animals,including humans.

Such agents may be formulated into compositions comprising an agenttogether with a pharmaceutically acceptable carrier or diluent. Theagent may in the form of a physiologically functional derivative, suchas an ester or a salt, such as an acid addition salt or basic metalsalt, or an N or S oxide. Compositions may be formulated for anysuitable route and means of administration. Pharmaceutically acceptablecarriers or diluents include those used in formulations suitable fororal, rectal, nasal, inhalable, topical (including buccal andsublingual), vaginal or parenteral (including subcutaneous,intramuscular, intravenous, intradermal, intrathecal and epidural)administration. The choice of carrier or diluent will of course dependon the proposed route of administration, which, may depend on the agentand its therapeutic purpose. The formulations may conveniently bepresented in unit dosage form and may be prepared by any of the methodswell known in the art of pharmacy. Such methods include the step ofbringing into association the active ingredient with the carrier whichconstitutes one or more accessory ingredients. In general theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product.

For solid compositions, conventional non-toxic solid carriers include,for example, pharmaceutical grades of mannitol, lactose, cellulose,cellulose derivatives, starch, magnesium stearate, sodium saccharin,talcum, glucose, sucrose, magnesium carbonate, and the like may be used.The active compound as defined above may be formulated as suppositoriesusing, for example, polyalkylene glycols, acetylated triglycerides andthe like, as the carrier. Liquid pharmaceutically administrablecompositions can, for example, be prepared by dissolving, dispersing,etc, an active compound as defined above and optional pharmaceuticaladjuvants in a carrier, such as, for example, water, saline aqueousdextrose, glycerol, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of non-toxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like, for example, sodium acetate, sorbitan monolaurate,triethanolaminc sodium acetate, sorbitan monolaurate, triethanolarnineoleate, etc. Actual methods of preparing such dosage forms are known, orwill be apparent, to those skilled in this art; for example, see Gennaroet al., Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa., 18th Edition, 1990.

The composition or formulation to be administered will, in any event,contain a quantity of the active compound(s) in an amount effective toalleviate the symptoms of the subject being treated.

Dosage forms or compositions containing active ingredient in the rangeof 0.25 to 95% with the balance made up from non-toxic carrier may beprepared.

For oral administration, a pharmaceutically acceptable non-toxiccomposition is formed by the incorporation of any of the normallyemployed excipients, such as, for example, pharmaceutical grades ofmannitol, lactose, cellulose, cellulose derivatives, sodiumcrosscarmellose, starch, magnesium stearate, sodium saccharin, talcum,glucose, sucrose, magnesium, carbonate, and the like. Such compositionstake the form of solutions, suspensions, tablets, pills, capsules,powders, sustained release formulations and the like. Such compositionsmay contain 1%-95% active ingredient, more preferably 2-50%, mostpreferably 5-8%.

Parenteral administration is generally characterized by injection,either subcutaneously, intramuscularly or intravenously. Injectables canbe prepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution or suspension in liquidprior to injection, or as emulsions. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol or the like. Inaddition, if desired, the pharmaceutical compositions to be administeredmay also contain minor amounts of non-toxic auxiliary substances such aswetting or emulsifying agents, pH buffering agents and the like, such asfor example, sodium acetate, sorbitan monolaurate, triethanolamineoleate, triethanolamine sodium acetate, etc.

The percentage of active compound contained in such parentalcompositions is highly dependent on the specific nature thereof, as wellas the activity of the compound and the needs of the subject. However,percentages of active ingredient of 0.1% to 10% in solution areemployable, and will be higher if the composition is a solid which willbe subsequently diluted to the above percentages. Preferably, thecomposition will comprise 0.2-2% of the active agent in solution.

Throughout this description the terms “standard methods”, “standardprotocols” and “standard procedures”, when used in the context ofmolecular biology techniques, are to be understood as protocols andprocedures found in an ordinary laboratory manual such as: CurrentProtocols in Molecular Biology, editors F. Ausubel et al., John Wileyand Sons, Inc. 1994, or Sambrook, J., Fritsch, E. F. and Maniatis, T.,Molecular Cloning: A laboratory manual, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. 1989.

This invention will be better understood by reference to theExperimental Details that follow, but those skilled in the art willreadily appreciate that these are only illustrative of the invention asdescribed more fully in the claims that follow thereafter. Additionally,throughout this application, various publications are cited. Thedisclosure of these publications is hereby incorporated by referenceinto this application to describe more fully the state of the art towhich this invention pertains.

EXPERIMENTAL PART I Synthesis of GABA_(B) Agonists

In the procedures described hereinafter the following abbreviations wereused: “DIPE” stands for diisopropylether; “EtOAc” stands for ethylacetate.

For some chemicals the chemical formula was used, e.g. CH₃CN foracetonitrile, NH₃ for ammonia, CH₂Cl₂ for dichloromethane, MgSO₄ formagnesium sulfate, and HCl for hydrochloric acid.

A. Preparation of the Intermediates

EXAMPLE A.1

Preparation of

4Aminobutanoic acid 1,1-dimethylethyl ester [50479-22-61 (14 g, 0.087mol) and 5,5-dimethyl-1,3-cyclohexanedione [126-81-8] (12.26 g, 0.087mol) were dissolved in trichloromethane (250 ml) andN,N-diethylethanamine (0.5 ml) was added. The reaction mixture wasstirred for 3 days and subsequently washed with three portions of 250 mlof water. The organic layer was dried on MgSO₄ and concentrated underreduced pressure. The residue was recrystallised in DIPE/CH₃CN to give18.6 g (76%) of intermediate 1.

This product was taken up in methanol (250 ml) and water (100 ml).1-Bromo-2,5-pyrrolidinedione (1 1.8 g, 0.066 mol) was added portionwiseover a 30 minutes period. After stirring for an additional hour, 500 mlwater was added The mixture was extracted with three portions ofdichloromethane. The combined organic layers were dried on MgSO₄ andconcenterated under reduced pressure to yield 22 g (92%) of intermediate2.

In a similar way was also prepared:

EXAMPLE A.2

Preparation of

A mixture of 5,6-diamino-4(1H)-pyrimidinethione [2846-89-1](0.0027 mol)and intermediate 2 (0.0027 mol) in ethanol (q.s.) was stirred for 2hours at 85° C. The reaction mixture was filtered and the solvent wasevaporated. The residue was purified by high-performance liquidchromatography. The product fractions were collected and the solvent(CH₃CN) was evaporated. The aqueous layer was extracted with EtOAc. Theorganic layer was separated, dried (MgSO₄), filtered and the solvent wasevaporated, yielding 0.400 g (30%) of intermediate 4.

EXAMPLE A.3

Preparation of intermediate

A mixture of 2-arninobenzcncthiol [137-07-5] (0.004 mol) andintermediate 2 (0.004 mol) in 1-methyl-2-pyrrolidinone [872-504] (15 ml)was stirred for 1 hour at 140° C. The reaction mixture was cooled andthe layers were separated with EtOAc/H₂O(NH₃). The organic layer wasdried (MgSO₄), filtered and the solvent was evaporated. The residue waspurified by high-performance liquid chrornatography. The productfractions were collected and the solvent (CH₃CN) was evaporated. Theaqueous layer was extracted with EtOAc and then the organic layer wasdried (MgSO₄), filtered off and the solvent was evaporated, yielding 0.6g (40%) of intermediate 5.

B. Prenaration of the Compounds

EXAMPLE B.1

Preparation of

A mixture of intermediate 4 (0.001 mol) in trifluoroacetic acid (5 ml)and dichloromethane (5 ml) was stirred for 1 hour at room temperature.The reaction mixture was dried under a stream of nitrogen. The resultingresidue was suspended in diethyl ether. The desired product was filteredoff and dried (vacuo) at 30° C., yielding 0.120 g (23%) oftrifluoroacetic acid salt of compound 2.

In a similar way were also prepared:

The hydrobromic acid salt of

and the trifluoroacetic acid salt of

EXAMPLE B.2

Preparation of

A mixture of intermediate 5 (0.00155 mol) in trifluoroacetic acid (5 ml)and dichloromethane (5 ml) was stirred for 20 hours at room temperature.The reaction mixture was dried under a stream of nitrogen. The resultingresidue was solidified in diethyl ether. The desired product wasfiltered off and dried (vacuo) at 30° C., yielding 0.320 g (67%) oftrifluoroacetic acid salt of compound 3.

II DEVELOPMENT OF GABA_(B)-CHO-K1 CELLS Material and Methods

Pennanent transfection of GABA_(B)R1a and GABA_(B)R2 in CHO-K1 cellsusing Lipofectamine PLUS:

CHO-cells were transfected with hGABABR1a/pcDNA3.1. Monoclonal stableR1a-expressing cells were transfected with hGABABR2/pcDNA3.1 Hygro+.Selection of clones occurred with 800 μg geneticin+800 μghygromycine/ml.

Menibrane Preparation:

Butyrate-stimulated (5 mM final) cells were scraped, after a short rinsewith PBS, in 50 mM TrisHCl pH7.4 and centrifuged at 23500 g for 10 min.at 4° C. The pellet was homogenised in 5 mM TrisHCI pH 7.4 byUltra-Turrax (24000 rpm) followed by centrifugation at 30000 g for 20min. at 4° C. The resulting pellet was resuspended in 50 mM TrisHCl pH7.4 and rehomogenised. Protein concentration was determined using theBradford method.

GTPγ35S Activation Assay:

10 μg membrane prep was incubated in 250 μl in 20 mM Hepes pH 7.4, 100mM NaCl, 3 mM MgCl2, 0.25 nM GTPγ35S, 3 μM GDP, 10 μg saponin/ml, withor without 1 mM GABA (basal activity in absence of baclofen) at 37° C.for 20 min. Filtration was carried out onto 96-well GF/B filter plate inHarvester (Packard). Filters were rinsed 6 times with cold 10 mMphosphate buffer pH 7.4, and dried overnight before addition of 30 μlMicroscint O, and measurement in Topcount (Packard, 1 min./well).

3H-Agonist Binding:

30-60 μg membrane prep was incubated in 50 mM TrisHCI pH 7.4, 2.5 mMCaCl2, 10 nM 3H-GABA or 20 nM 3H-baclofen in 500 μl at 20° C.Non-specific binding was determined in the presence of 100 μM baclofen.After 90 minutes the mixture was transferred onto 96-well GF/Bfilterplate by Harvester (Packard). Filters were rinsed 6 times withcold 50 mM TrisHCl pH 7.4, 2.5 mM CaCl2, and dried overnight beforeaddition of 30 μl Microscint O, and measurement in Topcount (Packard, 1min./well).

Results

GTPγ³⁵S Activation Assay

In membranes of stably hGABABR1a-transfected CHO-cells, we measuredbinding of the antagonist 3H-CGP54626. hGABABR2 was co-transfected inthose R1a-clones with the highest antagonist binding. After subcloningstable clones were obtained showing functional activity inGTPγ35S-binding assay upon stimulation of membranes by GABA, whereinsaid activity was potentiated in the presence of the positive modulatorCGP7930 (Urwyler S., et al., 2001, Molecular Pharmacology60:963-971)(FIG. 1).

Agonist Filter Binding Assay

An agonist filter binding assay has been developed in 96-well GF/Bfilterplate. The IC50 of known agonists and antagonists was determined(FIG. 2). While the stable hGABA_(B)R1a or the transient hGABA_(B)R2monomeric GABA_(B) receptor expressing cells did not show any binding tothe agonists 3H-GABA or 3H-baclofen (data not shown), unexpectedly, inour hGABABR1a/R2 beterodimeric clone agonist binding was detected withboth ligands. The Kd for 3H-baclofen, 3H-GABA, and 3H-CGP54626 wasdetermined in saturation experiments and compared well with publishedresults obtained with tissue preparations (table 1). TABLE 1 ³H-baclofenRat 132 nM (Hill & Bowery, 1981) Dog cortex 28 nM (J&JPRD, 2000)hGABA_(B)R1aR2/CHO 30 nM (our data, n = 2)) ³H-GABA Rat 77 nM (Hill &Bowery, 1983) Rat 15-30 nM (Cross & Horton, 1988) Pig 26 nM (Facklam &Bowery, 1993) Human 20-30 nM (Cross & Horton, 1988) hGABA_(B)R1aR2/CHO10-30 nM (our data, n = 6) ³H-CGP54626 Rat 1.5 nM (Bittiger et al.,1993) Pig 1.35 nM (Facklam & Bowery, 1993) hGABA_(B)R1aR2/CHO 1.5 nM(Green et al., 1993) hGABA_(B)R1aR2/CHO 2.78 nM (our data, n = 1)

The order of potency for agonists was AMPA>GABA>baclofen, and forantagonists CGP54626>SCH50911 (FIG. 2). The obtained IC50s werereproducible between different membrane preparations (FIG. 3)

Upon full library screening we identified some compounds with bindingand signal transduction properties with comparable potencies as thereference compounds GABA and baclofen (table 2). BINDING ASSAY SIGNALTRANSDUCTION ³H-GABA binding GTPγS binding Chemistry pIC50 % Effect at10 μM Reference compounds

6.90775 75.7021

8.06026 79.2906 HTS hits

7.1875 45.7275

6.82 40.95

6.43 24.44

6.87 61.93

Table 2: pIC₅₀ and % effect in the GABA ligand binding, and GTPγS signaltransduction assays for reference compounds and HTS hits.

Agonist centrifugation Binding AssayIn an alternative binding assay thenon-bound ligand was separated from the membranes by centrifugationinstead of filtration. The assay was performed according to the earlierdescribed filter binding assay, with the difference that the non-boundligand was separated from the membranes by centrifugation in amicrocentrifuge at 12500 rpm for 10 minutes. The supernatant wasdiscarded, the pellet was rinsed with washing buffer and dissolved in200 μl water. Scintillation fluid was added and the bound ³H-GABAmeasured in Topcount (Packhard, 1 min./well).

In a saturation assay using increasing concentrations of ³H-GABA (1-400nM final) I was found that the GABA_(B) receptor expressed by thehGABA_(B)R1a/GABA_(B)R2 CHO cell line, possess a low and a high affinityagonist binding site. Results of the saturation and scatchard analysisare summarized in Table 3. When the saturation assay was preformed inthe presence of 10 μM of the GABA_(B) antagonist CGP54626 or one of theGABA_(B) agonist of the present invention (compound 1), the ³H-GABAbinding to both the high and the low affinity site was blocked (FIGS. 4a, b). TABLE 3 Mean (n = 5) SD nM nM Bmax 1 0.19 0.05 Kd 1 9.4 3.1 Bmax2 0.76 0.24 Kd 2 401 224

Discussion

To our knowledge, no earlier reports were made in literature ofrecombinant hGABA_(B) receptor, showing agonist binding with a high andlow affinity binding site in a filter binding assay. An HTS agonistfilter binding screen has been developed using 3H-GABA. We foundreproducible Ki values for known agonists and antagonists, independentof the membrane preparation.

It has in addition been demonstrated that the recombinant GABA_(B)receptor has two agonist binding sites. One high affinity and one lowaffinity binding site. It is to be expected that high affinity agonistsof the GABA_(B) receptor will ellict a different response compared tothe low affinity agonists. Hence, the cell line of the present inventionnot only allows to identify GABA_(B) receptor agonists, but alsoprovides a useful tool to characterize the nature of the compoundreceptor interaction.

1. An isolated GABA_(B) receptor protein comprising at least oneGABA_(B)R1a subunit and at least one GABA_(B)R2 subunit, characterizedin that said GABA_(B) receptor has one high affinity agonist bindingsite and one low affinity agonist binding site.
 2. The GABA_(B) receptorprotein according to claim 1 wherein the GABA_(B)R1a subunit is encodedby the oligonucleotide sequence consisting of SEQ ID No.1 and theGABA_(B)R2 subunit is encoded by the oligonucleotide sequence consistingof SEQ ID NO.3.
 3. The GABA_(B) receptor protein according to claim 1wherein said receptor protein is expressed by thehGABA_(B)R1a/GABA_(B)R2 CHO cell line deposited at the BelgianCoordinated Collections of Microorganisms (BCCM) as CHO-K1 h-GABA-b R1a/R2 clone 20 on Aug. 22, 2003 with the accession number LMBP 6046CB. 4.Use of the GABA_(B) receptor protein according to claim 1 in a method toidentify GABA_(B) receptor agonists or antagonists.
 5. ThehGABA_(B)R1a/GABA_(B)R2 CHO cell line deposited at the BelgianCoordinated Collections of Microorganisms (BCCM) as CHO-K1 h-GABA-bR1a/R2 clone on Aug. 22, 2003 with the accession number LMBP 6046CB. 6.A method to identify whether a test compound binds to a GABA_(B)receptor protein according to claim 1, and is thus a potential agonistor antagonist of the GABA_(B) receptor, said method comprising: a)contacting cells expressing a functional GABA_(B) receptor, wherein suchcells do not normally express the GABA_(B) receptor, with the testcompound in the presence and absence of a compound know to bind to theGABA_(B) receptor, and b) determine the binding of the test compound tothe GABA_(B) receptor using the compound known to bind to the GABA_(B)receptor as a reference.
 7. A method according to claim 6, wherein thecompound known to bind to the GABA_(B) receptor is detectably labeled,and wherein said label is used to determine the binding of the testcompound to the GABA_(B) receptor.
 8. A method according to claim 7wherein the compound known to bind to the GABA_(B) receptor is selectedfrom the group consisting of ³H-GABA, ³H-baclofen, ³H-3-APPA,3H-CGP542626 and ³H-SCH50911.
 9. A method to identify GABA_(B) receptoragonists said method comprising, a) exposing cells expressing afunctional GABA_(B) receptor, wherein such cells do not normally expressthe GABA_(B) receptor, to a labeled agonist of GABA_(B) in the presenceand absence of the test compound, and b) determine the binding of thelabeled agonist to said cells, where if the amount of binding of thelabeled agonist is less in the presence of the test compound, then thecompound is a potential agonist of the GABA_(B) receptor.
 10. A methodaccording to claim 10 wherein the labeled agonist is selected from thegroup consisting of ³H-GABA, ³H-baclofen and ³H-3-APPA.
 11. A method toidentify GABA_(B) receptor antagonists said method comprising, a)exposing cells expressing a functional GABA_(B) receptor, wherein suchcells do not normally express the GABA_(B) receptor, to a labeledantagonist of GABA_(B) in the presence and absence of the test compound,and b) determine the binding of the labeled antagonist to said cells,where if the amount of binding of the labeled antagonist is less in thepresence of the test compound, then the compound is a potentialantagonist of the GABA_(B) receptor.
 12. A method according to claim 10wherein the labeled antagonist is selected from the group consisting of³H-CGP542626 and ³H-SCH50911.
 13. A method for identifying a compound asa GABA_(B) receptor agonist, said method comprising; a) administeringthe compound to a cellular composition of the cells according to claim5, in the presence of a detectably labeled GABA_(B) receptor agonist;and b) determine the binding of the labeled agonist to said cellularcomposition, where if the amount of binding of the labeled agonist isless in the presence of the test compound, then the compound is apotential agonist of the GABA_(B) receptor.
 14. A method according toclaim 13 wherein the cellular composition consists of a membranefraction of the hGABA_(B)R1a/GABA_(B)R2 CHO cell line deposited at theBelgian Coordinated Collections of Microorganisms (BCCM) as CHO-K1h-GABA-b R1a/R2 clone on Aug.
 22. 2003 with the accession number LMBP6046CB.
 15. A method according to claim 13 wherein the labelled agonistis selected from the group consisting of ³H-GABA, ³H-baclofen and³H-3-APPA.
 16. A method for identifying a compound as a GABA_(B)receptor antagonist, said method comprising; a) administering thecompound to a cellular compositon of the cells according to claim 5, inthe presence of a detectably labeled GABA_(B) receptor antagonist; andb) determine the binding of the labeled antagonist to said cellularcomposition, where if the amount of binding of the labeled antagonist isless in the presence of the test compound, then the compound is apotential antagonist of the GABA_(B) receptor.
 17. A method according toclaim 16 wherein the cellular composition consists of a membranefraction of the hGABA_(B)R1a/GABA_(B)R2 CHO cell line deposited at theBelgian Coordinated Collections of Microorganisms (BCCM) as CHO-K1h-GABA-b R1a/R2 clone on Aug. 22, 2003 with the accession number LMBP6046CB.
 18. A method according to claim 16 wherein the labeledantagonist is selected from the group consisting of ³H-CGP542626 and³H-SCH50911.
 19. A method for identifying compounds that have thecapability to modulate GABA_(B) receptor activity, said methodcomprising; a) contacting cells expressing a functional GABA_(B)receptor, wherein said cells do not normally express a functionalGABA_(B) receptor, with at least one reference compound, underconditions permitting the activation of the GABA_(B) receptor; b)contacting the cells of step a) with a test compound, under conditionspermitting the activation of the GABA_(B) receptor, and c) determinewhether said test compound modulates the GABA_(B) receptor activitycompared to the reference compound.
 20. A method according to claim 19wherein the capability of the test compound to modulate the GABA_(B)receptor activity is determined using one or more of the functionalresponses selected form the group consisting of changes in potassiumcurrents, changes in calcium concentration, changes in cAMP and changesin GTPγS binding
 21. A method for identifying compounds that have thecapability to modulate GABA_(B) receptor activity, said methodcomprising; a) contacting a membrane fraction of the cells according toclaim 5, with the compound to be tested in the presence of radiolabeldGTPγS, under conditions permitting the activation of the GABA_(B)receptor; and b) determine GTPγS binding to the membrane fraction, wherean increase in GTPγS binding in the presence of the compound is anindicaton that the compound activates the GABA_(B) receptor activity.22. A method for identifying compounds that have the capability tomodulate GABA_(B) receptor activity, said method comprising; a)contacting a membrane fraction of the cells according to claim 5, withthe compound to be tested in the presence of radiolabeld GTPγS, underconditions permitting the activation of the GABA_(B) receptor; and b)determine GTPγS binding to the membrane fraction, where an decrease inGTPγS binding in the presence of the compound is an indicaton that thecompound inactivates the GABA_(B) receptor activity.
 23. A methodaccording to claim 21 wherein the conditions permitting the activationof the GABA_(B) receptor comprise the presence of a GABA_(B) receptoragonist.
 24. A method according to claim 23 wherein the GABA_(B)receptor agonist is selected from the group consisting of GABA, baclofenand 3-APPA.
 25. Use of a compounds of formula (I)

the N-oxide forms, the pharmaceutically acceptable addition salts andthe stereochemically isomeric forms thereof, wherein;=Z¹-Z²=Z³-Z⁴=represents a divalent radical selected from the groupconsisting of ═N—CH═CH—N═ (a), ═N—CH═N—CH═ (b), ═CH—N═CH—N═ (c)═CH—CH═CH—CH═ (d), ═N—CH═CH—CH═ (e), ═CH—N═CH—CH═ (f), ═CH—CH═N—CH═ (g)and ═CH—CH═CH—N═ (h); R¹ represents hydrogen, halo, hydroxyl, cyano,C₁₋₆alkyl, CF₃, amino or mono- or di(C₁₋₄alkyl)amino; R² representshydrogen, C₁₋₆alkyl or hydroxycarbonyl-C₁₋₆alkyl-, in the manufacture ofa medicament for the treatment of an indication such as stiff mansyndrome, gastroesophogeal reflux, neuropathic pain, incontinence andtreatment of cough and cocaine addiction.
 26. Use of a compound offormula (I) in the manufacture of a medicament to reduce transient loweresophagal sphincter relaxations (TLESR).
 27. A compound of formula (I)wherein =Z¹-Z²=Z³-Z⁴=represents (a), (b) or (d), more preferably thosecompounds of formula (I) wherein =Z¹-Z²=Z³Z4=represents (d).
 28. Acompound according to claim 27 for use as a medicine.
 29. Use of acompound according to claim 27 in the manufacture of a medicament toreduce transient lower esophagal sphincter relaxations (TLESR).